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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/06—Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide

Definitions

• the invention relates to a method for the decontamination of mycoplasma-containing cell cultures.
• a first step cell cultures infected with mycoplasma are subjected to antibiotic treatment, and in a second step cells that are still infected are then eliminated by 6-methylpurine deoxyribose solution.
• Mycoplasmas are bacteria-like microorganisms that do not synthesize a cell wall or a mucous capsule. They multiply both in cell cultures (cytoadsorbed) and in cell-free medium. Characteristic of mycoplasma is the need for cholesterol or other steroid compounds.
• the largest sources of infection are contaminated sera and already infected cell cultures.
• the transmission is usually via droplet infection or aerosols.
• An infected culture contains between 106-109 colony forming units (CFU) per milliliter of conditioned medium without the medium becoming cloudy or having a characteristic odor.
• CFU colony forming units
• mycoplasma strains leading to degenerative effects in cell culture due to extensive acid production.
• the most common strains of mycoplasma such as M. arginini, M. orale, M. hyorhinis, and A. laidlawii, proliferate without observing morphological changes in the contaminated cell line.
• chromosomal aberrations and altered cell metabolism are often found in mycoplasma-contaminated cultures.
• This cytotoxicity is based on the enzyme adenosine phosphorylase, which is synthesized in mycoplasmas, but not in animal cells.
• 6-methylpurine deoxyribose (6-MPDR) or 6-methylpurine ribose (6-MPR) are cleaved, with 6-methylpurine (6-MP) being released as the substance toxic to the cells (GJ McGarrity, et al (1982), Exp. Cell Research, 139, 199).
• 6-methylpurine (6-MP) 6-methylpurine (6-MP) being released as the substance toxic to the cells.
• the result is a lysis of the cell culture contaminated with mycoplasma, since concentrations of less than 5 ⁇ M of the cleavage product 6-MP are toxic to the cells. Because all relevant mycoplasma strains Containing adenosine phosphorylase, this test system also detects all possible sources of contamination.
• the invention consequently relates to a method for the decontamination of cell cultures containing mycoplasma, antibiotics effective against mycoplasmas being used in a first step.
• mycoplasma-containing cells or cell clones are then eliminated by cultivation in the presence of 6-methylpurine deoxyribose, so that only the cells or cell clones that are actually free of mycoplasmas remain, ie survive.
• Thiamulin and minocycline are preferably used as antibiotics and 6-methylpurine deoxyribose is used in a concentration of 50 ⁇ M.
• the invention further relates to the mycoplasma-free cell cultures obtained in this way and to their use, in particular in the genetic engineering production of foreign proteins in these cells.
• the invention is additionally disclosed in the claims and the example.
• Rodent cells represent a preferred expression system for the expression of heterologous recombinant proteins.
• CHO (chinese hamster ovary) and BHK (baby hamster kidney) cells are most frequently used. Both cell lines are established cultures and are already used for the large-scale production of recombinant proteins.
• BHK and CHO clones that secreted a specific protein were isolated using a complex transfection, selection and cloning process. Mycoplasma contamination was detected in some of these clones.
• the contaminated cell cultures were initially treated alternately with 2 passages each with the antibiotics thiamulin and minocycline (Sebio, concentration according to the manufacturer's instructions). The cells were then kept 4 passages completely without antibiotics. After checking in the mycoplasma test, the mycoplasma-free cultures were isolated from clones. For this purpose, the cells were sown via "limiting dilution" in a cell density of 2 cells / well and 10 cells / well on a 96-hole dish. The creation of 5 wells per dilution per culture had proven to be sufficient.
• the cells were only treated with the antibiotics described above, isolated from clones and not kept in the presence of 6-MPDR. The cells were then frozen and partially kept in culture for control purposes. Mycoplasma tests were also carried out after 5, 10, 15 and 20 passages, mycoplasma again being detectable in approximately 30% of the cultures after passage 10. The cells were kept under conditions under which a new infection with mycoplasma could be excluded.
• This method was also used with the same success in human cell cultures (Hela and KB cells) and in monkey cells (COS cells).

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• Health & Medical Sciences (AREA)
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• Genetics & Genomics (AREA)
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• Microbiology (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Cell Biology (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Agricultural Chemicals And Associated Chemicals (AREA)

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Citations (3)

• 1989
  • 1989-05-26 DE DE3917163A patent/DE3917163A1/de active Granted
• 1990
  • 1990-05-24 PT PT94145A patent/PT94145B/pt not_active IP Right Cessation
  • 1990-05-24 AU AU55900/90A patent/AU617052B2/en not_active Ceased
  • 1990-05-24 KR KR1019900007513A patent/KR100220988B1/ko not_active IP Right Cessation
  • 1990-05-25 JP JP2134256A patent/JP2934482B2/ja not_active Expired - Lifetime
  • 1990-05-25 IE IE189390A patent/IE66910B1/en not_active IP Right Cessation
  • 1990-05-25 ES ES90109939T patent/ES2072331T3/es not_active Expired - Lifetime
  • 1990-05-25 EP EP90109939A patent/EP0399545B1/fr not_active Expired - Lifetime
  • 1990-05-25 CA CA002017568A patent/CA2017568A1/fr not_active Abandoned
  • 1990-05-25 DE DE59008875T patent/DE59008875D1/de not_active Expired - Fee Related
  • 1990-05-25 DK DK90109939.0T patent/DK0399545T3/da active
  • 1990-05-25 AT AT90109939T patent/ATE121131T1/de not_active IP Right Cessation
• 1993
  • 1993-02-03 US US08/012,985 patent/US5902740A/en not_active Expired - Fee Related

Patent Citations (3)

Non-Patent Citations (2)

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